LOCUS       sequence-80654-e        6215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA precursor with CspCI placeholder for target 
            cloning (see comments)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6215)
  AUTHORS   Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M
  TITLE     Implementation of the CRISPR-Cas9 system in fission yeast.
  JOURNAL   Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344.
  PUBMED    25352017
REFERENCE   2  (bases 1 to 6215)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2014-10-29"; volume: "5:5344. doi"; pages: " 
            10.1038/ncomms6344"
COMMENT     Sequence Label: pMZ283
FEATURES             Location/Qualifiers
     source          1..6215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      539..1320
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     CDS             1930..2724
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     primer_bind     3350..3372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3364..3381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3365..3381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(3431..3482)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(3601..3957)
                     /label=rrk1 promoter/leader
                     /note="promoter and leader RNA for the Schizosaccharomyces 
                     pombe rrk1 gene encoding the K-RNA component of RNase P 
                     (Jacobs et al., 2014)"
     primer_bind     complement(3994..4010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3994..4010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4007..4029)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4018..4034
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4042..4048,4049..4066,4067..4072))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4087..4108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4225..4242)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4396..4984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4476..4495)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5155..5946,5947..6015))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5778..5797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6016..6120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6188..6206
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataatcg atggccaacc ttccaattca ttaaatcgag tcctccatca gatgatttac
      241 aaggttttgc atagaatccc taagtaagag aagttgttag tgatttcttc aagtttacta
      301 aaatttacat acaaatggat gatctcttct ccattgtttc ctttcttctt gcaaacgcgt
      361 tttacaaaga gatgacatgc tattttctga ttattttttt ctatagtttt ctattttgta
      421 cgtccttggg agtccgaaat gtaaaatcgg ggtatggtta gtagcggcag cgttcattat
      481 ggtatataaa agataaaaag taggggcaag cgagacagaa tgggatacaa gggcatcgtc
      541 tataattata gctaaaaatt gtattttaat ttgtattttt tgtaatttta tttctcattg
      601 ttttacttaa aatgaatgcg aattagagaa aacttcaacg aaatgtcaaa ataagctcag
      661 caaaatatac aattttaggg aaagcgatca gcaacttatt ctccgttgta gattcttttt
      721 cttcataaat tccaacaatg taactgccaa ttgatttttc tctagatgct aataaattga
      781 aactaatgaa tataataaca caggttttga taaattgtaa actcttactc aaaaaaagaa
      841 tgttaaaact aatttaacct ttttataaag gctcatggcg ttaaagagga tagtaaaaat
      901 tctacaattt tttaatttaa tgtcacaatt gtcttgcgaa tataattcta ttaattttcg
      961 ctaactgctg ttttagtact attactttca atatgcctta tatgcaactt taattcatga
     1021 agaatgcaat atttttgcaa tctttcttgt actgagcgct ttttatcatt tagtactcat
     1081 tatttaattt ttcagtaaag ggcaataaat aaatttgtag aagatgcaat gtaatctcct
     1141 ctatcctttt gctcatatgt ttatgagtat acctagtcta gaaaggcttg tatttaaaat
     1201 attattcaat aaaaattcac aatttttacg acatgtgcta ccattcactt aacttcctga
     1261 ttataaaatt ggttcgttta tactaattac ttaagtacct ttaactaaac aaaatgccta
     1321 tatatatatt aatttacaat gagtgtcaga taagtcacta tgtccgagtg gttaaggagt
     1381 tagactcgcc atcgatgagc ttagctacaa atcccactgg ctatatgtat gcatttgtgt
     1441 taaaaaagtt tgtatagatt atttaatcta ctcagcattc tttctctaaa taggaatttg
     1501 ttacttaatg gagaaaaaaa tgtttcgatt tacctagtgt atttgtttgt atactcacgt
     1561 ttaatttcaa acatccattc tatcttgtgt aatttttggc atggtgaaaa agataatcag
     1621 ccttataatc tttacaaaag taagaaattc tgtaaataag ccttaatgcc cttgctttaa
     1681 attaaaatgg ttctttttca tgataatgtt tgcactttgt gaatatattt tagatagttc
     1741 tgtgaggtat aattaagatg ttttagagac ttatacaatt ttgtctttat aaattcttaa
     1801 ttgattttac catcccagtt taactatgct tcgtcggcat ctctgcacat gtcgtgtttt
     1861 cttaccgtat tgtcctacca agaacctctt ttttgcttgg atcgaaatta aaggtttaaa
     1921 agcaaagtta tggatgctag agtatttcaa agctattcag ctagagctga ggggatgaaa
     1981 aatcccattg ccaaggaatt gttggctttg atggaagaaa agcaaagcaa cttgtcagtc
     2041 gcggtcgatt tgacgaagaa atccgaaatc ttagaattgg tagataaaat tggaccctat
     2101 gtctgtgtta tcaagacaca tattgacgtt gtcgaggatt tcgaccagga tatggtagaa
     2161 aaactggtgg ccttaggtaa aaagcatcgt tttcttatct ttgaggatcg caaattcgca
     2221 gacattggaa ataccgtcaa gctacaatat gcatctggtg tgtacaaaat tgcttcttgg
     2281 gctcatatca caaattgcca tacagtgcca ggcgagggta ttatacaagg cctcaaagaa
     2341 gttggtttac ctttgggacg tggtctcttg cttttggctg aaatgtcttc caaaggctct
     2401 ttggctactg gttcctacac agagaaaacc ttagaatggt ttgagaagca taccgatttt
     2461 tgctttggct ttatagctgg tcgtcgattt cctaaccttc aaagcgacta cataactatg
     2521 tcccctggta tcggcttgga tgttaaagga gacgggctgg gacagcaata tcgtactcct
     2581 gaagaagtga ttgtaaactg cggtagcgat atcatcattg ttggtcgtgg agtctatgga
     2641 gctggtcgta atcctgttgt cgaagccaag agatatagag aagctggttg gaaggcatat
     2701 cagcaaagac tttctcagca ttaaaaaaag actaatgtaa aatttttttg gttggttatt
     2761 gaaaaagtcg atgccttgtt tgcgtttgtt ttcctaggcg ttttatgtca gaaggcattt
     2821 agaattagta tacaagtact ctttggtaaa attttatgta gcgactaaaa tattaactat
     2881 tatagataaa caccttggga ataaaaagta atttgctata gtaatttatt aaacatgctc
     2941 ctacaacatt accacaatct tttctcttgg attgacattg aataagaaaa gagtgaattt
     3001 ttttagactt gtaatgataa ctatgtacaa agccaatgaa agatgtatgt agatgaatgt
     3061 aaaataccat gtagacaaac aagataaaac ttggttataa acattggtgt tggaacagaa
     3121 taaattagat gtcaaaaagt ttcgtcaata tcacaagctc atcgcgatgg tatgcggtgt
     3181 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     3241 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     3301 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     3361 cgttgtaaaa cgacggccag tgccaagctt gcatgcctgc aggtcgactc tagaggatcc
     3421 ccacggtacc cctgtttcgt cctcacggac tcatcagacc ggaaagcaca tccggtgaca
     3481 gggcaccacc gactcggtgc cactcacttt tttcaagttg ataacggact agccttattt
     3541 taacttgcta tttctagctc taaaacagag agataaccca caagaattga gttaagccca
     3601 ttcttcggta caggttatgt tttttggcaa cagcaactat tcaaatcaac tgaaagtttt
     3661 ttagaattca acaacttcca atactcaagt gtccaagcta gcaaactgtt taataatcgt
     3721 gacccaacat tcctttcatg cgcaccctca gcgtcacata aatcaacact ttacaacacc
     3781 tattcaaagc ttctatatgc cttcgtcatt taaatgtcta acatttcaaa aaagtgtttg
     3841 ctaatggtaa attcttgagt tttcttggaa gcacatatat aacgagtgtg actgttctac
     3901 gctccttgct gactagtcat cagtctacgc atgccaacta ccaccaacat aagcaaaagg
     3961 tacccggggt accgagctcg aattcgtaat catgtcatag ctgtttcctg tgtgaaattg
     4021 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4081 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4141 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4201 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4261 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4321 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4381 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4441 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4501 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4561 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4621 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4681 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4741 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4801 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4861 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4921 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4981 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5041 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5101 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5161 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5221 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5281 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5341 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5401 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5461 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5521 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5581 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5641 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5701 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5761 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5821 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5881 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5941 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6001 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6061 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6121 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6181 ctataaaaat aggcgtatca cgaggccctt tcgtc
//
